This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our laboratory has evolved a family of non-biological ATP binding proteins that have been evolved from a pool of purely random sequences. The crystal structure of a representative of one of these families was recently solved and shown to bind ATP/ADP, and a divalent Zn2+ ion. Additional rounds of directed evolution, designed to select for stronger ligand binding and structural stability, yielded a consensus sequence which differed from its progenitor by only 2 amino acids, was shown to display both ATP/ADP binding, possessed much higher affinity for substrate, and was shown to be much more resistant to denaturation in the presence of GuHCl. Co-crystallization studies carried out in the presence of varying amounts of ATP have revealed a variety of new ligand binding modes. We expect the contribution of the 2 amino acid substitutions to be discreet, yet significant in their contribution to the new structural and biochemical observations. To test this hypothesis, we would like to carry out MAD experiments at the zinc absorption edge (peak, remote, and inflection) to acquire unbiased and independent phases for each construct and their individual point mutants to determine the structural and functional implications these 2 mutations have in our novel, non-biological ATP binding protein. These studies promise useful information into how nature has selected for proteins that have developed the ability for specialized ligand binding, but have also evolved the capability of catalyzing specific enzymatic reactions on that ligand. In addition, these studies should allow us to glean important insight into the necessary parameters required (i.e. protein architecture, ligand binding and catalytic scaffolds, etc.) for design of new, and potentially better, ligand binders, and enzymes.